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Address to: Dr. Dionatas Ulises de Oliveira Meneguetti. PPGBIOEXP/UNIR. Campus-BR 364, Km 9.5, 76801-059 Porto Velho, RO, Brasil.Phone: 55 69 9243-7860e-mail: dionatas@icbusp.orgReceived 7 September 2013Accepted 27 November 2013

First report of Rhodnius montenegrensis (Hemiptera: Reduviidae: Triatominae) infection

by Trypanosoma rangeliDionatas Ulises de Oliveira Meneguetti[1], Evanildo Bezerra Soares[2],

Marta Campaner[3] and Luis Marcelo Aranha Camargo[4],[5]

[1]. Programa de Pós Graduação em Biologia Experimental, Fundação Universidade Federal de Rondônia, Porto Velho, RO. [2]. Departamento de Saúde, Faculdade de Educação e Meio Ambiente, Ariquemes, RO. [3]. Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP. [4]. Instituto de Ciências Biomédicas, Universidade de São Paulo, Monte Negro, RO. [5]. Departamento de Medicina, Faculdade São Lucas, Porto Velho, RO.

ABSTRACTIntroduction: This study reports for the fi rst time the infection of Rhodnius montenegrensis by Trypanosoma rangeli. Methods: The triatomines were manually collected in Attalea speciosa in the municipality of Buritis, Rondônia. The identifi cation of the trypanosomatid species was confi rmed by multiplex PCR. Results: All of the collected triatomines were R. montenegrensis. The analysis confi rmed that all of the adults were infected with the epimastigote form of T. rangeli. Conclusions: This report of a new vector of T. rangeli raises a warning for the State of Rondônia because the simultaneous presence of T. rangeli with T. cruzi in the same geographic region enables the occurrence of mixed infections in hosts and vectors, which complicates the differential diagnosis.

Keywords: Triatominae. Trypanosoma rangeli. Rhodnius montenegrensis. Western Amazon.

Trypanosoma rangeli is a heterogeneous hemofl agellate protozoan discovered in Venezuela in the intestinal contents of Rhodnius prolixus and, in addition to Trypanosoma cruzi, is the only trypanosome parasite of man found in Central and South America1.

Trypanosoma rangeli has been reported in Colombia, Mexico, Guatemala, Honduras, El Salvador, Nicaragua, Costa Rica, Panama, Guyana, Fr Guyana, Uruguay, Paraguay, Peru, Chile, Venezuela, Trinity, Argentina, Ecuador, Bolivia and Brazil2,3. In Brazil, it was found in wild animals and triatomines in the south (Santa Catarina), southeast (Minas Gerais), northeast (Ceará, Alagoas and Bahia), central west (Federal District, Goiás, Mato Grosso and Mato Grosso do Sul) and north (Amazonas, Pará, Rondônia, Acre and Tocantins) regions2,4,5.

From the earliest reports of human infection by T. rangeli in the Americas, more than 2,700 cases of human rangeliosis have been confi rmed6. The only cases of human infection reported in Brazil have been in the States of Amazonas and Bahia7,8, but in contrast to T. cruzi, T. rangeli is not considered pathogenic to its vertebrate hosts3.

The transmission of T. rangeli to vertebrates is mainly related to species of triatomines of the genus Rhodnius, with 12 species reported as their natural vectors; of these 12 species, 6 are found

in Brazil (Rhodnius neglectus, Rhodnius nasutus, Rhodnius neivai, Rhodnius domesticus, Rhodnius pictipes and Rhodnius robustus)2,9,10. The latter species may have been erroneously described in the State of Rondônia by some researchers11,12 because of its similarity to R. montenegrensis, which had not been described at that time.

Rhodnius montenegrensis was fi rst described in 2012 from specimens collected in the municipality of Monte Negro, Rondônia, Brazil. Initially, it was identifi ed as R. robustus, but subsequent studies showed that it was a new species13.

The present study reports the fi rst documented infection of Rhodnius montenegrensis (Hemiptera: Reduviidae: Triatominae) by Trypanosoma rangeli.

Monthly collections were performed with permission from the Brazilian Institute of Environment and Renewable Natural Resources (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis - IBAMA), permanent license No. 14934-1 from June to December of 2012 in the rural pasture area in the municipality of Buritis, Rondônia (Latitude 9º57’32.3”S - Longitude 64º08’05.2”W) (Figure 1).

Triatomines were collected from 14 specimens of Attalea speciosa (babassu) twice a month in the morning after downing a tree with a chainsaw and removing the bracts. Triatomines were manually collected through active searching. The A. speciosa were randomly selected, drawing any 1 from a group of 20.

The collected specimens were sent in a cooler at room temperature to the microscopy laboratory at the School of Education and Environment (Faculdade de Educação e Meio Ambiente - FAEMA) in the municipality of Ariquemes, Rondônia, where wet mount analysis and smear analysis of

Revista da Sociedade Brasileira de Medicina Tropical 47(3):374-376, May-Jun, 2014http://dx.doi.org/10.1590/0037-8682-0179-2013Short Communication

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Meneguetti DUO et al - Rhodnius montenegrensis: a new vector of Trypanosoma rangeli

AmazonasAmazonas

Bolivia

*

The State of Rondônia

Northern - Brazil BoliviaN

N

NW NE

SW SE

L

S

W

0 100 200 300 400km

Mato Grosso

* Buritis

MatoGro

sso

FIGURE 1 - Geographical location of the municipality of Buritis, State of Rondônia, Brazil.

the adult triatomines’ rectal contents were performed with an optical microscope at 1600X magnifi cation after staining with triarylmethane (0.1%), xanthene (0.1%) and thiazine (0.1%) (Figure 2).

The identification of the trypanosomatid species was confi rmed by multiplex polymerase chain reaction (PCR) in collaboration with the Department of Parasitology at the Institute of Biomedical Sciences, São Paulo University (ICB-USP), São Paulo (SP), Brazil.

Parasite deoxyribonucleic acid (DNA) was extracted from the triatomine rectal samples using a Qiagen DNA extraction kit. The multiplex PCR was performed according to Fernandes et al.14. This method amplifi es a portion of the non-transcribed spacer of the mini-exon gene that varies between T. cruzi and T. rangeli species and between lines 1 and 2 of T. cruzi. The following primers were used: TC1, 5’-ACACTTTCTGGCGCTGATCG-3’; TC2, 250bp, 5’-TTGCTCGCACACTCGGCTGCAT-3’; Z3, 150bp, 5’-CCGCGCACAACCCCTATAAAAATG-3’; TR, 100bp, 5’-CCTATTGTGATCCCCATCTTCG-3’ and EXON, 5’-TACCAATATAGTACAGAACTG-3’. The reaction mixture consisted of 100pmol of each primer and 150μM dNTPs in a buffer composed of 10mM Tris-HCl (pH 8.3), 1.5mM MgCl2,

25mM KCl, 0.1mg/ml bovine serum albumin, 2.5U of Taq DNA polymerase and 10ng of genomic DNA in a total volume of 50μL. The thermal cycling conditions were as follows: an initial step of 5min at 95°C, 34 cycles of 30sec at 94°C, 30sec at 55°C and 30sec at 72°C and a fi nal extension of 10min at 72°C. The following reference strains were used as controls in each reaction: TC1, X10 Clone 1; TC2, Strain Y; Z3, Emerald Clone 1 and T. rangeli R1625. The amplifi ed products were subjected to electrophoresis on a 2% agarose gel at 100V for 1h. After electrophoresis, the DNA was stained with ethidium bromide and visualized under ultraviolet light. A molecular marker of 50 base pairs was used as a size control for the amplifi ed fragments.

The identification of the adult triatomine species was conducted based on the keys previously described by and Rosa et al.12.

A total of 120 triatomines were collected, providing an average of 8.6 specimens per Attalea speciosa, which is below the average reported by other studies in the state9,11. Of those triatomines, only 13 (10.8%) were adults, and all were R. montenegrensis.

The analysis confi rmed that all of the adults were infected with trypanosomatids, with the T. rangeli in the epimastigote form.

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Rev Soc Bras Med Trop 47(3):374-376, May-Jun, 2014

The authors declare that there is no confl ict of interest.

CONFLICT OF INTEREST

REFERENCES

This work is the fi rst report of the infection of R. montenegrensis by T. rangeli, which increases the total number of species of triatomines of the genus Rhodnius infected by this protozoan to 13, including 7 species found specifi cally in Brazil.

This report of a new vector of T. rangeli presents a warning to the State of Rondônia because the simultaneous presence of T. rangeli and T. cruzi in the same geographic region enables the occurrence of mixed infections in both vertebrate hosts and vectors, which complicates the differential diagnosis of an infection15. This possibility is concerning, especially in a state where there are reports of other vector species of T. rangeli, such as R. pictipes and R. robustus11,12.

FIGURE 2 - Epimastigote form of Trypanosoma rangeli at 1600X magnifi cation.

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